Novel mutations in human mlh1 and human msh2 genes useful in diagnosing colorectal cancer

ABSTRACT

Variant human MLH1 and MSH2 genes are provided. Methods of using these variant genes to diagnose hereditary non-polyposis colorectal cancer (HNPCC) and/or determine a patient&#39;s susceptibility to developing HNPCC are also provided. Methods and compositions for identifying new variant MLH1 of MSH2 genes are also provided. In addition, experimental models for hereditary non-polyposis colorectal cancer comprising these variant genes are provided.

[0001] This application claims the benefit of U.S. provisional application Ser. No. 60/105,180, filed Oct. 22, 1998.

BACKGROUND OF THE INVENTION

[0002] Colorectal cancer (CRC) is one of the most common fatal cancers in developed countries, and the worldwide incidence is increasing. The United States and the United Kingdom are high incidence countries, with an estimated 133,500 new cases and 55,300 deaths (Parker et al. CA Cancer J. Clin. 1996 46:5-27) in the United States and 30,941 cases and approximately 17,000 deaths in the United Kingdom (HMSO UK Cancer Registry Data). The population lifetime risk is 1 in 25 in the United States and Northern Europe and thus represents a significant public health issue (Sharp et al. Cancer Registration Statistics Scotland 1981-1990, Information and Statistics Division, The National Health Service in Scotland, Edinburgh (1993)). Identification of people who are predisposed to the disease would allow targeting of effective preventative measures with the aim of reducing the considerable cancer related mortality (Burke et al. J. Am. Med. Ass'n. 1997 227:915-919).

[0003] One group of people with a very high colorectal cancer risk are those who carry germline mutations in genes that participate in DNA mismatch repair. hMSH2 (Fishel et al. Cell 1993 75:1027-1038; Leach et al. Cell 1993 75:1215-1225; U.S. Pat. No. 5,591,826) and hMLH1 (Bronner et al. Nature 1994 368:258-261; Papadopoulos et al. Science 1994 263:1625-1629; PCT Publication No. WO 95/20678, published on Aug. 3, 1995) are the two genes most commonly involved in heredity predisposition to CRC, but mutations in hPMS1 and hPMS2 also occur in a minority of cases (Nicolaides et al. Nature 1994 371:75-80). Such mutations are usually associated with marked familial aggregation of colorectal, uterine and other cancers constituting the clinically defined autosomal dominant syndrome of hereditary non-polyposis colorectal cancer (HNPCC) (Lynch et al. Gastroenterology 1993 104:1535-1549; Liu et al. Nature Med. 1996 2:169-174; Wijnen et al. Am. J. Hum. Genet. 1995 56:1060-1066; Mary et al. Hum. Mol. Genet. 1994 3:2067-2069; Nystrom-Lahti et al. Nature Med. 1995 1:1203-1206). However, an appreciable proportion of patients who have early onset colorectal cancer but who do not fulfill pragmatic criteria for HNPCC (Vasen et al. Dis. Colon Rectum 1991 34:424-425) also carry mismatch repair gene mutations (Liu et al. Nature Med. 1995 2:169-174; Dunlop et al. Br. Med. J. 1997 314:1779-1780). Thus, restricting genetic testing to individuals from families fulfilling HNPCC criteria is likely to exclude a significant fraction of gene carriers in the general population. However, screening unselected patients with sporadic cancer represents an enormous workload and may provide a very low yield of mutation carriers (Liu et al. Nat. Med. 1995 1:348-352; Tomlinson et al. J. Med. Genet. 1997 34:39-42).

[0004] It is clear that issues concerning indications for genetic testing and interpretation of results are critical in hereditary cancer syndromes (Giardiello et al. N. Engl. J. Med. 1997 336: 823-827).

[0005] Using a population-based approach, factors indicative of the likelihood of identifying patients with mismatch repair gene mutations were investigated. Improved approaches to mutation detection and the prevalence of detectable mismatch repair gene alterations in various screened groups who were not selected on the basis of family history were also determined.

SUMMARY OF THE INVENTION

[0006] An object of the present invention is to provide novel, variant hMLH1 sequences.

[0007] Another object of the present invention is to provide novel, variant hMSH2 sequences.

[0008] Another object of the present invention is to provide a method of diagnosing hereditary non-polyposis colorectal cancer in a patient or determining a patient's susceptibility to developing hereditary non-polyposis colorectal cancer via detection of novel variant hMLH1 or hMSH2 sequences or the exonic or intronic sequences of the hMLH1 and hMSH2 genes.

[0009] Another object of this invention is to provide methods and compositions for identifying new variants of hMLH1 and hMSH2 genes.

[0010] Yet another object of the present invention is to provide experimental models of hereditary non-polyposis colorectal cancer.

DETAILED DESCRIPTION OF THE INVENTION

[0011] To better elucidate the structure of human MLH1 and human MSH2 genes and to determine possible sites of alternative splicing, the genes were cloned and sequenced and PCR was used to determine alternate splice products (variants) and exon/intron boundaries. Elucidation of intron/exon boundary sequences revealed that hMLH1 is encoded by 19 coding exons. The hMLH1 gene sequence was determined by PCR.

[0012] The intron/exon structure of the hMLH1 is shown below. Positions of introns that interrupt the hMLH1 cDNA are shown. Exonic sequence is presented in upper case and intronic sequence in lower case letters. Exons are numbered from the 5′ end of the cDNA sequence.

[0013] hMLH1 Exon 1

[0014] aggcactgaggtgattggc (SEQ ID NO:1)

[0015] tgaaggcacttccgttgagcatctagacgtttccttggctcttctggcgccaaa (SEQ ID NO:2)

[0016] ATGTCGTTCGTGGCAGGGGTTATTCGGCGGCTGGACGAGACAGTGGTGAACCGCATCGC GGCGGGGGAAGTTATCCAGCGGCCAGCTAATGCTATCAAAGAGATGATTGAGAACTG (SEQ ID NO:3)

[0017] gtacggagggagtcgagccgg (SEQ ID NO:4)

[0018] gctcacttaagggctacga (SEQ ID NO:5)

[0019] cttaacgg (SEQ ID NO:6)

[0020] hMLH1 Exon 2

[0021] aatatgtacattagagtagttg (SEQ ID NO:7)

[0022] cagactgataaattattttctgtttgatttgccag (SEQ ID NO:8)

[0023] TTTAGATGCAAAATCCACAAGTATTCAAGTGATTGTTAAAGAGGGAGGCCTGAAGTTGA TTCAGATCCAAGACAATGGCACCGGGATCAGG (SEQ ID NO:9)

[0024] gtaagtaaaacctcaaagtagcaggatgtttgtgcgcttcatggaa (SEQ ID NO:10)

[0025] gagtcaggacctttctctg (SEQ ID NO:11)

[0026] hMLH1 Exon 3

[0027] agagatttggaaaatgagtaac (SEQ ID NO:12)

[0028] atgattatttactcatctttttggtatctaacag (SEQ ID NO:13)

[0029] AAAGAAGATCTGGATATTGTATGTGAAAGGTTCACTACTAGTAAACTGCAGTC CTTTGAGGATTTAGCCAGTATTTCTACCTATGGCTTTCGAGGTGAG (SEQ ID NO:14)

[0030] gtaagctaaagattcaagaaatgtgtaaaatat (SEQ ID NO:15)

[0031] cctcctgtgatgacattgt (SEQ ID NO:16)

[0032] c

[0033] hMLH1 Exon 4

[0034] aacctttccctttggtgagg (SEQ ID NO:17)

[0035] tgacagtgggtgacccagcagtgagtttttctttcagtctattttcttttcttcttag (SEQ ID NO:18)

[0036] GCTTTGGCCAGCATAAGCCATGTGGCTCATGTTACTATTACAACGAAAACAGCT GATGGAAAGTGTGCATACAG (SEQ ID NO:19)

[0037] gtatagtgctgacttcttttactcatatatattcattctgaaatgtattttgg (SEQ ID NO:20)

[0038] gcctaggtctcagagtaatc (SEQ ID NO:21)

[0039] hMLH1 Exon 5

[0040] ttgatat (SEQ ID NO: 22)

[0041] gattttctcttttccccttggg (SEQ ID NO:23)

[0042] attagtatctatctctctactggatattaatttgttatattttctcattag (SEQ ID NO: 24)

[0043] AGCAAGTTACTCAGATGGAAAACTGAAAGCCCCTCCTAAACCATGTGCTGGCAATCAAG GGACCCAGATCACG (SEQ ID NO: 25)

[0044] gtaagaatggtacatgggaca (SEQ ID NO:26)

[0045] gtaaattgttgaagctttgtttg (SEQ ID NO:27)

[0046] hMLH1 Exon 6

[0047] gggttttattttcaagtacttctatg (SEQ ID NO: 28)

[0048] aatttacaagaaaaatcaatcttctgttcag (SEQ ID NO: 29)

[0049] GTGGAGGACCTTTTTTACAACATAGCCACGAGGAGAAAAGCTTTAAAAAATCCAAGT GAAGAATATGGGAAAATTTTGGAAGTTGTTGGCAG (SEQ ID NO:30)

[0050] gtacagtccaaaatctgggagtgggtctctgagatttgtcatcaaagtaatgtgttctagt (SEQ ID NO:31)

[0051] gctcatacattgaacagttgctgagc (SEQ ID NO:32)

[0052] hMLH1 Exon 7

[0053] ctagtgtgtgtttttggc (SEQ ID NO:33)

[0054] aactcttttcttactcttttgtttttcttttccag (SEQ ID NO:34)

[0055] GTATTCAGTACACAATGCAGGCATTAGTTTCTCAGTTAAAAAA (SEQ ID NO:35)

[0056] gtaagttcttggtttatgggggatggttttgttttatgaaaagaaaaaaggggattttt aatagtttgct (SEQ ID NO:36)

[0057] ggtggagataaggttatg (SEQ ID NO:37)

[0058] hMLH1 Exon 8

[0059] ctcagccatgagacaataaatcc (SEQ ID NO:38)

[0060] ttgtgtcttctgctgtttgtttatcag (SEQ ID NO:39)

[0061] CAAGGAGAGACAGTAGCTGATGTTAGGACACTACCCAATGCCTCAACCGTGGACAATAT TCGCTCCATCTTTGGAAATGCTGTTAGTCG (SEQ ID NO:40)

[0062] gtatgtcgataacctatataaaaaaatcttttacatttattatcttggtttatcatt (SEQ ID NO:41)

[0063] ccatcacattatttgggaacc (SEQ ID NO: 42)

[0064] hMLH1 Exon 9

[0065] caaaagcttcagaatctc (SEQ ID NO: 43)

[0066] ttttctaatag (SEQ ID NO:44)

[0067] AGAACTGATAGAAATTGGATGTGAGGATAAAACCCTAGCCTTCAAAATGAATGGTTACA TATCCAATGCAAACTACTCAGTGAAGAAGTGCATCTTCTTACTCTTCATCAACC (SEQ ID NO:45)

[0068] gtaagttaaaaagaaccacatgggaaat (SEQ ID NO:46)

[0069] ccactcacaggaaacacccacag (SEQ ID NO:47)

[0070] hMLH1 Exon 10

[0071] catgactttgtgtgaatgtacacc (SEQ ID NO: 48)

[0072] tgtgacctcacccctcaggacagttttgaactggttgctttctttttattgtttag (SEQ ID NO:49)

[0073] ATCGTCTGGTAGAATCAACTTCCTTGAGAAAAGCCATAGAAACAGTGTATGCAGCCTATT TGCCCAAAAACACACACCCATTCCTGTACCTCAG (SEQ ID NO:50)

[0074] gtaatgtagcaccaaactcctcaaccaagactcacaaggaa (SEQ ID NO:51)

[0075] cagatgttctatcaggctctcctc (SEQ ID NO: 52)

[0076] hMLH1 Exon 11

[0077] gggctttttctccccctccc (SEQ ID NO:53)

[0078] actatctaaggtaattgttctctcttattttcctgacag (SEQ ID NO: 54)

[0079] TTTAGAAATCAGTCCCCAGAATGTGGATGTTAATGTGCACCCCACAAAGCATGAAG TTCACTTCCTGCACGAGGAGAGCATCCTGGAGCGGGTGCAGCAGCACATCGAGAGCAAG CTCCTGGGCTCCAATTCCTCCAGGATGTACTTCACCCAG (SEQ ID NO:55)

[0080] gtcagggcgcttctcatccagctacttctctggggcctttgaaatgtgcccggccaga (SEQ ID NO:56)

[0081] cgtgagagcccagatttt (SEQ ID NO:57)

[0082] hMLH1 Exon 12

[0083] aattatacctcatactagc (SEQ ID NO:58)

[0084] ttctttcttagtactgctccatttggggacctgtatatctatacttcttattctgagtct ctccactatatatatatatatatatatatttttttttttttttttttttaatacag (SEQ ID NO:59)

[0085] ACTTTGCTACCAGGACTTGCTGGCCCCTCTGGGGAGATGGTTAAATCCACAACAAGTCT GACCTCGTCTTCTACTTCTGGAAGTAGTGATAAGGTCTATGCCCACCAGATGGTTCGTA CAGATTCCCGGGAACAGAAGCTTGATGCATTTCTGCAGCCTCTGAGCAAACCCC TGTCCAGTCAGCCCCAGGCCATTGTCACAGAGGATAAGACAGATATTTCTAGTGGCAGGG CTAGGCAGCAAGATGAGGAGATGCTTGAACTCCCAGCCCCTGCTGAAGTGGCTGCCAAAA ATCAGAGCTTGGAGGGGGATACAACAAAGGGGACTTCAGAAATGTCAGAGAAGAGAGGAC CTACTTCCAGCAACCCCAG (SEQ ID NO:60)

[0086] gtatggccttttgggaaaagtacagccta (SEQ ID NO:61)

[0087] cctcctttattctgtaataaaac (SEQ ID NO:62)

[0088] hMLH1 Exon 13

[0089] tgcaacccacaaaatttggc (SEQ ID NO:63)

[0090] taagtttaaaaacaagaataataatgatctgcacttccttttcttcattgcag (SEQ ID NO:64)

[0091] AAAGAGACATCGGGAAGATTCTGATGTGGAAATGGTGGAAGATGATTCCCGAAAGGAAA TGACTGCAGCTTGTACCCCCCGGAGAAGGATCATTAACCTCACTAGTGTTTTGAGTCTCCAG GAAGAAATTAATGAGCAGGGACATGAGG (SEQ ID NO:65)

[0092] gtacgtaaacgctgtggcctgcctgggatgcatagggcctcaactgccaa (SEQ ID NO: 66)

[0093] ggttttggaaatggagaaag (SEQ ID NO:67)

[0094] hMLH1 Exon 14

[0095] tggtgtctctagttctgg (SEQ ID NO: 68)

[0096] tgcctggtgctttggtcaatgaagtggggttggtaggattctattacttacctgttttt tggttttattttttgttttgcag (SEQ ID NO:69)

[0097] TTCTCCGGGAGATGTTGCATAACCACTCCTTCGTGGGCTGTGTGAATCCTCAGTGGGCCTTG GCACAGCATCAAACCAAGTTATACCTTCTCAACACCACCAAGCTTAG (SEQ ID NO:70)

[0098] gtaaatcagctgagtgtgtgaacaa (SEQ ID NO:71)

[0099] gcagagctactacaacaatg (SEQ ID NO: 72)

[0100] hMLH1 Exon 15

[0101] cccatttgtcccaactg9 SEQ ID NO:73

[0102] ttgtatctcaagcatgaattcagcttttccttaaagtcacttcatttttattttcag (SEQ ID NO:74)

[0103] TGAAGAACTGTTCTACCAGATACTCATTTATGATTTTGCCAATTTTGGTGTTC TCAGGTTATCG (SEQ ID NO:75)

[0104] gtaagtttagatccttttcactt (SEQ ID NO:76)

[0105] ctgacatttcaactgaccg (SEQ ID NO:77)

[0106] hMLH1 Exon 16

[0107] catttggatgctccgttaaagc (SEQ ID NO:78)

[0108] ttgctccttcatgttcttgcttcttcctag (SEQ ID NO:79)

[0109] GAGCCAGCACCGCTCTTTGACCTTGCCATGCTTGCCTTAGATAGTCCAGAGAGTGGCTG GACAGAGGAAGATGGTCCCAAAGAAGGACTTGCTGAATACATTGTTGAGTTTCTGAAGA AGAAGGCTGAGATGCTTGCAGACTATTTCTCTTTGGAAATTGATGAG (SEQ ID NO:80)

[0110] gtgtgacagccattcttatacttctgttgtattctc (SEQ ID NO:81) caaataaaatttccagccgggtg (SEQ ID NO:82)

[0111] hMLH1 Exon 17

[0112] ggaaaggcactggagaaatggg (SEQ ID NO:83)

[0113] atttgtttaaactatgacagcattatttcttgttcccttgtcctttttcctgcaagcag (SEQ ID NO:84)

[0114] GAAGGGAACCTGATTGGATTACCCCTTCTGATTGACAACTATGTGCCCCCTTTGGAGGG ACTGCCTATCTTCATTCTTCGACTAGCCACTGAG (SEQ ID NO:85)

[0115] gtcagtgatcaagcagatactaagcattt (SEQ ID NO:86)

[0116] cggtacatgcatgtgtgctggaggg (SEQ ID NO:87)

[0117] hMLH1 Exon 18

[0118] taagtagtctgtgatctccg (SEQ ID NO:88)

[0119] tttagaatgagaatgtttaaattcgtacctattttgaggtattgaatttctttggaccag (SEQ ID NO:89)

[0120] GTGAATTGGGACGAAGAAAAGGAATGTTTTGAAAGCCTCAGTAAAGAATGCGCTATGTT CTATTCCATCCGGAAGCAGTACATATCTGAGGAGTCGACCCTCTCAGGCCAGCAG (SEQ ID NO:90)

[0121] tacagtggtgatgcacactggcaccccaggacta (SEQ ID NO:91)

[0122] gacaggacctcatacat (SEQ ID NO:92)

[0123] MLH1 Exon 19

[0124] gacaccagtgtatgttgg (SEQ ID NO:93)

[0125] gatgcaaacagggaggcttatgacatctaatgtgttttccag (SEQ ID NO:94)

[0126] AGTGAAGTGCCTGGCTCCATTCCAAACTCCTGGAAGTGGACTGTGGAACACATTGTC TATAAAGCCTTGCGCTCACACATTCTGCCTCCTAAACATTTCACAGAAGATGGAAATATC CTGCAGCTTGCTAACCTGCCTGATCTATACAAAGTCTTTGAGAGGTGTTAA (SEQ ID NO:95)

[0127] atatggttatttatgcactgt (SEQ ID NO:96)

[0128] gggatgtgttcttctttctc (SEQ ID NO:97)

[0129] tgtattccgatacaaagtgttgtatcaaagtgtgatatacaaagtgtaccaacataagtg (SEQ ID NO:98)

[0130] Elucidation of intron/exon boundary sequences revealed that hMSH2 is encoded by 16 coding exons. The hMSH2 gene sequence was determined by PCR.

[0131] The intron/exon structure of the hMSH2 is shown below. Positions of introns that interrupt the hMSH2 cDNA are shown. Exonic sequence is presented in upper case and intronic sequence in lower case letters. Exons are numbered from the 5′ end of the cDNA sequence.

[0132] hMSH2 Exon 1

[0133] ggcgggaaacagcttagtgggtgtggggtcg (SEQ ID NO:99)

[0134] cgcattttcttcaaccagga (SEQ ID NO:100)

[0135] ggtgaggaggtttcgac (SEQ ID NO:101)

[0136] ATGGCGGTGCAGCCGAAGGAGACGCTGCAGTTGGAGAGCGCGGCCGAGGTCGGCTTCGTG CGCTTCTTTCAGGGCATGCCGGAGAAGCCGACCACCACAGTGCGCCTTTTCGACCGGGG CGACTTCTATACGGCGCACGGCGAGGACGCGCTGCTGGCCGCCCGGGAGGTGTTCAAGA CCCAGGGGGTGATCAAGTACATGGGGCCGGCAG (SEQ ID NO:102)

[0137] gtgagggccgggac (SEQ ID NO:103)

[0138] ggcgcgtgctggggagg (SEQ ID NO:104)

[0139] gac

[0140] hMSH2 Exon 2

[0141] gaa

[0142] gtccagctaatacagtgcttg (SEQ ID NO:105)

[0143] aacatgtaatatctcaaatctgtaatgtactttttttttttttaag (SEQ ID NO:106)

[0144] GAGCAAAGAATCTGCAGAGTGTTGTGCTTAGTAAAATGAATTTTGAATCTTTTGTAAAA GATCTTCTTCTGGTTCGTCAGTATAGAGTTGAAGTTTATAAGAATAGAGCTGGAAATAAG GCATCCAAGGAGAATGATTGGTATTTGGCATATAAG (SEQ ID NO:107)

[0145] gtaattatcttcctttttaatttacttattttt (SEQ ID NO:108)

[0146] ttaagagtagaaaaataaaaatgtg (SEQ ID NO:109)

[0147] aag

[0148] hMSH2 Exon 3

[0149] ATTAATAAGGtTCATAGAGTTTGGATTTTTCCtTTTtgc (SEQ ID NO:110)

[0150] ttataaaattttaaagtatgttcaag (SEQ ID NO:111)

[0151] agtttgttaaatttttaaaattttatttttacttag (SEQ ID NO:112)

[0152] GCTTCTCCTGGCAATCTCTCTCAGTTTGAAGACATTCTCTTTGGTAACAATGAT ATGTCAGCTTCCATTGGTGTTGTGGGTGTTAAAATGTCCGCAGTTGATGGCCAGAGACAG GTTGGAGTTGGGTATGTGGATTCCATACAGAGGAAACTAGGACTGTGTGAATTCCCTGAT AATGATCAGTTCTCCAATCTTGAGGCTCTCCTCATCCAGATTGGACCAAAGGAATGTGT TTTACCCGGAGGAGAGACTGCTGGAGACATGGGGAAACTGAGACAG (SEQ ID NO:113)

[0153] gtaagcaaattgagtctagtgat (SEQ ID NO:114)

[0154] agaggagattccaggcctaggaaag (SEQ ID NO:115)

[0155] gc

[0156] TCTTTAATTGACATGATACTG (SEQ ID NO:116)

[0157] hMSH2 Exon 4

[0158] ttca

[0159] tttttgcttttcttattccttttc (SEQ ID NO:117)

[0160] tcatagtagtttaaactatttctttcaaaatag (SEQ ID NO:118)

[0161] ATAATTCAAAGAGGAGGAATTCTGATCACAGAAAGARAAAAAGCTGACTTTTCCACAAA AGACATTTATCAGGACCTCAACCGGTTGTTGAAAGGCAAAAAGGGAGAGCAGATGAATA GTGCTGTATTGCCAGAAATGGAGAATCAG (SEQ ID NO:119)

[0162] gtacatggattataaatgtgaattacaatatatataatgtaaatatgtaatatataata aataatatgtaaactatagtgacttt (SEQ ID NO:120)

[0163] ttagaaggatatttctgtca (SEQ ID NO:121)

[0164] tat

[0165] hMSH2 Exon 5

[0166] actggcacca (SEQ ID NO:122)

[0167] gtggtatagaaatcttcgattttt (SEQ ID N0:123)

[0168] aaattcttaattttag (SEQ ID NO:124)

[0169] GTTGCAGTTTCATCACTGTCTGCGGTAATCAAGTTTTTAGAACTCTTATCAGATGATTC CAACTTTGGACAGTTTGAACTGACTACTTTTGACTTCAGCCAGTATATGAAATTGGATA TTGCAGCAGTCAGAGCCCTTAACCTTTTTCAG (SEQ ID NO:125)

[0170] gtaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO:126)

[0171] aaaagggttaaaaatgttgatt (SEQ ID NO:127)

[0172] gg

[0173] TTAAAAAATGTTT (SEQ ID NO:128)

[0174] t

[0175] caTTGACATATACTGAAGAAGCT (SEQ ID NO:129)

[0176] TATAAAGGAGCTAAAATATTTGGAAAT (SEQ ID NO:130)

[0177] att

[0178] ATTATACTTGGATTAGATAACTAGCTTTAAATGGGTGTATTTT (SEQ ID NO:131)

[0179] hMSH2 Exon 6

[0180] gtt

[0181] ttcactaatgagcttgccattc (SEQ ID NO:132)

[0182] tttctattttattttttgtttactag (SEQ ID NO:133)

[0183] GGTTCTGTTGAAGATACCACTGGCTCTCAGTCTCTGGCTGCCTTGCTGAATAAGTGTAA AACCCCTCAAGGACAAAGACTTGTTAACCAGTGGATTAAGCAGCCTCTCATGGATAAGA ACAGAATAGAGGAGAG (SEQ ID NO:134)

[0184] gtatgttattagtttatactttcgttagttttatgtaacctgca (SEQ ID NO:135)

[0185] gttacccacatgattatacc (SEQ ID NO:136)

[0186] ac

[0187] hMSH2 Exon 7

[0188] ga

[0189] cttacgtgcttagttgataa (SEQ ID NO:137)

[0190] attttaattttatactaaaatattttacattaattcaagttaatttatttcag (SEQ ID NO:138)

[0191] ATTGAATTTAGTGGAAGCTTTTGTAGAAGATGCAGAATTGAGGCAGACTTTACAAGAAG ATTTACTTCGTCGATTCCCAGATCTTAACCGACTTGCCAAGAAGTTTCAAAGACAAGCA GCAAACTTACAAGATTGTTACCGACTCTATCAGGGTATAAATCAACTACCTAATGTTAT ACAGGCTCTGGAAAAACATGAAG (SEQ ID NO:139)

[0192] gtaacaagtgattttgtttttttg (SEQ ID NO:140)

[0193] ttttccttcaactcatacaatata (SEQ ID NO:141)

[0194] tac

[0195] hMSH2 Exon 8

[0196] ga

[0197] tttgtattctgtaaaatgagatcttt (SEQ ID NO:142)

[0198] ttatttgtttgttttactactttcttttag (SEQ ID NO:143)

[0199] GAAAACACCAGAAATTATTGTTGGCAGTTTTTGTGACTCCTCTTACTGATCTTCGTTCT GACTTCTCCAAGTTTCAGGAAATGATAGAAACAACTTTAGATATGGATCAG (SEQ ID NO:144)

[0200] gtatgcaatatactttttaatttaag (SEQ ID NO:145)

[0201] cagtagttatttttaaaaagcaaag (SEQ ID NO:146)

[0202] gcc

[0203] hMSH2 Exon 9

[0204] gt

[0205] ctttacccattatttataggatt (SEQ ID NO:147)

[0206] ttgtcactttgttctgtttgcag (SEQ ID NO:148)

[0207] GTGGAAAACCATGAATTCCTTGTAAAACCTTCATTTGATCCTAATCTCAGTGAA TTAAGAGAAATAATGAATGACTTGGAAAAGAAGATGCAGTCAACATTAATAAGTGCAGC CAGAGATCTTG (SEQ ID NO:149)

[0208] gtaagaatgggtcattggag (SEQ ID NO:150)

[0209] gttggaataattcttttgtctat (SEQ ID NO:151)

[0210] ac

[0211] hMSH2 Exon 10

[0212] gg

[0213] tagtaggtatttatggaatactttt (SEQ ID NO:152)

[0214] tcttttcttcttgtttatcaag (SEQ ID NO:153)

[0215] GCTTGGACCCTGGCAAACAGATTAAACTGGATTCCAGTGCACAGTTTGGATATTACTTTC GTGTAACCTGTAAGGAAGAAAAAGTCCTTCGTAACAATAAAAACTTTAGTACTGTAGATA TCCAGAAGAATGGTGTTAAATTTACCAACAG (SEQ ID NO:154)

[0216] gtttgtaagtcattattatatttttaaccctttatt (SEQ ID NO:155)

[0217] aattccctaaatgctctaaca (SEQ ID NO:156)

[0218] tg

[0219] hMSH2 Exon 11

[0220] ca

[0221] cattgcttctagtacacattt (SEQ ID NO:157)

[0222] taatatttttaataaaactgttatttcgatttgcag (SEQ ID NO:158)

[0223] CAAATTGACTTCTTTAAATGAAGAGTATACCAAAAATAAAACAGAATATGAAGAAGCCC AGGATGCCATTGTTAAAGAAATTGTCAATATTTCTTCAG (SEQ ID NO:159)

[0224] gtaaacttaatagaactaa (SEQ ID NO:160)

[0225] taatgttctqaatgtcacctg (SEQ ID NO:161)

[0226] g

[0227] hMSH2 Exon 12

[0228] at

[0229] tcagtattcctgtgtacattt (SEQ ID NO:162)

[0230] tctgtttttatttttatacag (SEQ ID NO:163)

[0231] GCTATGTAGAACCAATGCAGACACTCAATGATGTGTTAGCTCAGCTAGATGCTGTTGTC AGCTTTGCTCACGTGTCAAATGGAGCACCTGTTCCATATGTACGACCAGCCATTTTGGAGAA AGGACAAGGAAGAATTATATTAAAAGCATCCAGGCATGCTTGTGTTGAAGTTCAAGATG AAATTGCATTTATTCCTAATGACGTATACTTTGAAAAAGATAAACAGATGTTCCACATC ATTACTG (SEQ ID NO:164)

[0232] gtaaaaaacctggttt (SEQ ID NO:165)

[0233] ttgggctttgtgggggtaa (SEQ ID NO:166)

[0234] cg

[0235] hMSH2 Exon 13

[0236] cg

[0237] cgattaatcatcagtgtac (SEQ ID NO:167)

[0238] agtttaggactaacaatccatttattagtagcagaaagaagtttaaaatcttgctttct gatataatttgttttgtag (SEQ ID NO:168)

[0239] GCCCCAATATGGGAGGTAAATCAACATATATTCGACAAACTGGGGTGATAGTACT CATGGCCCAAATTGGGTGTTTTGTGCCATGTGAGTCAGCAGAAGTGTCCATTGTGGACTG CATCTTAGCCCGAGTAGGGGCTGGTGACAGTCAATTGAAAGGAGTCTCCACGTTCATGGC TGAAATGTTGGAAACTGCTTCTATCCTCAG (SEQ ID NO:169)

[0240] gtaagtgcatctcctagtccctt (SEQ ID NO:170)

[0241] gaagatagaaatgtatgtctctg (SEQ ID NO:171)

[0242] tcc

[0243] hMSH2 Exon 14

[0244] ta

[0245] ccacattttatgtgatgggaa (SEQ ID NO:172)

[0246] atttcatgtaattatgtgcttcag (SEQ ID NO:173)

[0247] GTCTGCAACCAAAGATTCATTAATAATCATAGATGAATTGGGAAGAGGAACTTCTACCTA CGATGGATTTGGGTTAGCATGGGCTATATCAGAATACATTGCAACAAAGATTGGTGCTTT TTGCATGTTTGCAACCCATTTTCATGAACTTACTGCCTTGGCCAATCAGATACCAACTGT TAATAATCTACATGTCACAGCACTCACCACTGAAGAGACCTTAACTATGCTTTATCAGGT GAAGAAAG (SEQ ID NO:174)

[0248] gtatgtactattggagtactctaaattcagaacttg

[0249] gtaatgggaaacttactacc (SEQ ID NO:175)

[0250] cc

[0251] hMSH2 Exon 15

[0252] ct

[0253] cttctcatgctgtcccctc (SEQ ID NO:176)

[0254] acgcttccccaaatttcttatag (SEQ ID NO:177)

[0255] GTGTCTGTGATCAAAGTTTTGGGATTCATGTTGCAGAGCTTGCTAATTTCCCTAAGCAT GTAATAGAGTGTGCTAAACAGAAAGCCCTGGAACTTGAGGAGTTTCAGTATATTGGAGA ATCGCAAGGATATGATATCATGGAACCAGCAGCAAAGAAGTGCTATCTGGAAAGAGAG (SEQ ID NO:178)

[0256] gtttgtcagtttgtttt (SEQ ID NO:179)

[0257] catagtttaacttagcttctc (SEQ ID NO:180)

[0258] tat

[0259] hMSH2 Exon 16

[0260] ta

[0261] attactcatgggacattcaca (SEQ ID NO:181)

[0262] tgtgtttcag (SEQ ID NO:182)

[0263] CAAGGTGAAAAAATTATTCAGGAGTTCCTGTCCAAGGTGAAACAAATGCCCTTTAC TGAAATGTCAGAAGAAAACATCACAATAAAGTTAAAACAGCTAAAAGCTGAAGTAATAGC AAAGAATAATAGCTTTGTAAATGAAATCATTTCACGAATAAAAGTTACTACGTGA (SEQ ID NO:183)

[0264] aaa

[0265] atcccagtaatggaatgaag (SEQ ID NO:184)

[0266] gta

[0267] hMLH1 and hMSH2 genes were sequenced in 50 cancer patients (age of onset <30) and 26 random anonymous donors. Initial genomic sequencing detected 12 germline mutations in 12 patients (24%). Five mutations were found in hMLH1, and 7 in hMSH2. Using a combination of genomic sequencing and in vitro synthesized-protein-truncation assay (IVSP), a total of 15 germ-line mutations were identified. The mutations are described in Table 1.

[0268] Table 1: Pathogenic hMLH1 and hMSH2 Mutations Identified in Young Colorectal Cancer Probands TABLE 1 Pathogenic hMLH1 and hMSH2 Mutations Identified in Young Colorectal Cancer Probands Gene Effect on and Nucleotide Coding Patient Mutation Change Sequence Location hMLH1:  329 616delAAG Deletion of Deletion of Exon 16 AAG at 1846- Lys616 1848  533 IVS8- Deletion of Splice IVS 8 3delTA TA at 677-3 mutation  696 K618A AA→GC at Lys→Ala at Exon 16 1852-1853 618  804 R659X C→T at 1975 Arg→Stop at Exon 17 659  815 IVS1 + 1G→A G→A at 116 + 1 Splice IVS 1 mutation  817 del exon Deletion of Deletion of IVS 12- 13 ˜3 kb codons 470- 13, exon involving IVS 520 (exon 13 12 through 13) exon 13 to IVS 13  889 not Truncation Exons identified of IVSF 12-19 hMSH2:  528 R406X C→T at 1216 Arg→Stop at Exon 7 406  579 H639Y C→T at 1915 Double Exon 12, IVS13-1G→T G→T at 2211 mutation IVS 13 results in deletion of codons 588- 820 (exons 12-14)  814 Q601X C→T at 1801 Gln→Stop at Exon 12 601  818 Q252X C→T at 754 Gln→Stop at Exon 4 252  825 delCTGT Deletion of Deletion of Exon 5 CTGT at 808- codons 265- 811 314 (exon 5)  830 R680X C→T at 2038 Arg→Stop at Exon 13 680 1157 M1L A→T at 1 New Exon 1 initiation at codon 26

[0269] Two of the mutations identified in Table 1 for hMLH1 and three of the mutations identified in Table 1 for hMSH2 are believed to be new. For hMLH1, these include: the splice mutation IVS1+1G-A in patient 815, also referred to herein as “hMLH1 mutant 1”; and deletion of exon 13 in patient 817, also referred to herein as “hMLH1 mutant 2”. For hMSH2, these include the double mutation H639Y IVS13-1G-T leading to deletion of codons 588-820 in patient 579, also referred to herein as “hMSH2 mutant 1”, mutation R680X in patient 830 which comprises a nucleotide change from C to T at position 2038 in Exon 13 and results in a stop codon at position 680 of the coding sequence, also referred to herein as “hMSH2 mutant 2”; and mutation MIL in patient 1157 which comprises a nucleotide change from A to T at position 1 resulting in a -new initiation at codon 26, also referred to herein as “hMSH2 mutant 3”. Detection of these genetic mutations is useful in diagnosing HNPCC in a patient and determining susceptibility of a patient for developing HNPCC.

[0270] There are several methodologies available from recombinant DNA technology which may be used for detecting these new variants and identifying additional genetic mutations responsible for colon cancer. The identification of intronic sequences of hMLH1 and hMSH2 provided herein is particularly useful for design of intronic such as those exemplified in SEQ ID NO:1, 5, 7, 11, 12, 16, 17, 21, 23, 27, 28, 32, 33, 37, 38, 42, 43, 47, 48, 52, 53, 57, 58, 62, 63, 67, 68, 72, 73, 77, 78, 82, 83, 87, 88, 92, 93, 97, 100, 104, 105, 109, 111, 115, 117, 121, 123, 121, 123, 127, 129, 132, 136, 137, 141, 142, 146, 147, 151, 152, 156, 157, 161, 162, 166, 167, 171, 172, 175, 176, 180, 181 and 184 for use in identifying mutants in the splice donor or acceptor sites of the hMLH1 or hMSH2 gene. Examples of methodologies useful in detecting and identifying new variants of these genes include, but are not limited to, direct probing, ligase chain reaction (LCR) and polymerase chain reaction (PCR) methodology.

[0271] Detection of variants or mutants using direct probing involves the use of oligonucleotide probes which may be prepared synthetically or by nick translation. In a preferred embodiment, the probes are complementary to at least a portion of the variant hMLH1 or hMSH2 genes identified herein. The DNA probes may be suitably labeled using, for example, a radiolabel, enzyme label, fluorescent label, or biotin-avidin label, for subsequent visualization in for example a Southern blot hybridization procedure. The labeled probe is reacted with a sample of DNA from a patients suspected of having HNPCC bound to nitrocellulose or Nylon 66 substrate. The areas that carry DNA sequences complementary to the labeled DNA probe become labeled themselves as a consequence of the reannealing reaction. The areas of the filter that exhibit such labeling may then be visualized, for example, by autoradiography.

[0272] Alternative probe techniques, such as ligase chain reaction (LCR) involve the use of a mismatch probe, i.e., probes which have full complementarity with the target except at the point of the mutation or variation. The target sequence is then allowed to hybridize both with the oligonucleotides having full complementarity, i.e., oligonucleotides complementary to the hMLH1 or hMSH2 variants of the present invention, and oligonucleotides containing a mismatch under conditions which will distinguish between the two. By manipulating the reaction conditions, it is possible to obtain hybridization only where there is full complementarity. If a mismatch is present, then there is significantly reduced hybridization.

[0273] The polymerase chain reaction (PCR) is a technique that amplifies specific DNA sequences. Repeated cycles of denaturation, primer annealing and extension carried out with a heat stable enzyme Taq polymerase leads to exponential increases in the concentration of desired DNA sequences.

[0274] Given the knowledge of nucleotide sequences encoding the hMLH1 and hMSH2 genes, it is possible to prepare synthetic oligonucleotides complementary to the sequences which flank the DNA of interest. Each oligonucleotide is complementary to one of the two strands. The DNA is then denatured at high temperatures (e.g., 95° C.) and then reannealed in the presence of a large molar excess of oligonucleotides. The oligonucleotides, oriented with their 3′ ends pointing towards each other, hybridize to opposite strands of the target sequence and prime enzymatic extension along the nucleic acid template in the presence of the four deoxyribonucleotide triphosphates. The end product is then denatured again for another cycle. After this three-step cycle has been repeated several times, amplification of a DNA segment by more than one million fold can be achieved. The resulting DNA may then be directly sequenced in order to locate any genetic alterations. Alternatively, the identified hMLH1 and hMSH2 variants of the present invention make it possible to prepare oligonucleotides that will only bind to altered DNA, so that PCR will only result in the multiplication of the DNA if the mutation is present. Following PCR, allele-specific oligonucleotide hybridization may be used to detect the colon cancer point mutation.

[0275] Alternatively, an adaptation of PCR called amplification of specific alleles (PASA) can be employed; this method uses differential amplification for rapid and reliable distinction between alleles that differ at a single base pair. Newton et al. Nucleic Acid Res. 1989 17:2503; Nichols et al. Genomics 1989 5:535; Okayama et al. J. Lab. Clin. Med. 1989 1214:105; Sarkar et al. Anal. Biochem. 1990 186:64; Sommer et al. Mayo Clin. Proc. 1989 64:1361; Wu, Proc. Nat'l Acad. Sci. USA 1989 86:2757; and Dutton et al. Biotechniques 1991 11:700. PASA involves amplification with two oligonucleotide primers such that one is allele specific. The desired allele is efficiently amplified, while the other allele(s) is poorly amplified because it mismatches with a base at or near the 3′ end of the allele specific primer. Thus, PASA or the related method PAMSA can be used to specifically amplify one or more mutant hMLH1 or hMSH2 alleles. Where such amplification is performed on genetic material obtained from a patient, it can serve as a method of detecting the presence of one or more mutant hMLH1 and/or hMSH2 alleles in a patient- PCR-induced mutation restriction analysis, often referred to as IMPA, can also be used in the detection of mutants.

[0276] Also important is the development of experimental models of HNPCC. Such models can be used to screen for agents that alter the degenerative course of HNPCC. Having identified specific mutations in the hMLH1 and hMSH2 genes as a cause of HNPCC, it is possible using genetic manipulation, to develop transgenic model systems and/or whole cell systems containing a mutated hMLH1 and/or hMSH2 gene or a portion thereof. The model systems can be used for screening drugs and evaluating the efficacy of drugs in treating HNPCC. In addition, these model systems provide a tool for defining the underlying biochemistry of hMLH1 and hMSH2 and their relationship to HNPCC, thereby providing a basis for rational drug design.

[0277] One type of cell system which can be used in the present invention can be naturally derived. For this, blood samples from an affected individual are obtained and permanently transformed into a lymphoblastoid cell line using, for example, Epstein-Barr virus. Once established, such cell lines can be grown continuously in suspension cultures and can be used in a variety of in vitro experiments to study hMLH1 and hMSH2 expression and processing. Another cell line used in these studies comprises skin fibroblasts derived from patients.

[0278] The mutated gene can also be excised for use in the creation of transgenic animals containing the mutated gene. For example, the hMLH1 and hMSH2 variants of the present invention can each be cloned and placed in a cloning vector. Examples of cloning vectors which can be used include, but are not limited to, lCharon35, cosmid, or yeast artificial chromosome. The variant hMLH1 or hMSH2 gene can then be transferred to a host nonhuman knockout animal such as a knockout mouse. As a result of the transfer, the resultant transgenic nonhuman animal will preferably express one or more of the variant hMLH1 or hMSH2 polypeptides.

[0279] Alternatively, minigenes encoding variant hMLH1 or hMSH2 polypeptides can be designed. Such minigenes may contain a cDNA sequence encoding a variant hMLH1 or hMSH2 polypeptide, preferably full-length, a combination of hMLH1 or hMSH2 exons, or a combination thereof, linked to a downstream polyadenylation signal sequence and an upstream promoter (and preferably enhancer). Such a minigene construct will, when introduced into an appropriate transgenic host, such as a mouse or rat, express a variant hMLH1 or hMSH2 polypeptide.

[0280] One approach to creating transgenic animals is to target a mutation to the desired gene by homologous recombination in an embryonic stem (ES) cell in vitro followed by microinjection of the modified ES cell line into a host blastocyst and subsequent incubation in a foster mother. Frohman et al. Cell 1989 56:145. Alternatively, the technique of microinjection of the mutated gene, or portion thereof, into a one-cell embryo followed by incubation in a foster mother can be used. Additional methods for producing transgenic animals are well known in the art.

[0281] Transgenic animals are used in the assessment of new therapeutic compositions and in carcinogenicity testing, as exemplified by U.S. Pat. No. 5,223,610. These animals are also used in the development of predictive animal models for human disease states, as exemplified in U.S. Pat. No. 5,221,778. Therefore, the novel mutations of the hMLH1 and hMSH2 genes of the present invention, which are believed to cause HNPCC, provide a useful means for developing knockout transgenic animals to assess this disease.

[0282] Site directed mutagenesis and/or gene conversion can also be used to a mutate a non human hMLH1 or hMSH2 gene allele, either endogenously or via transfection, such that the mutated gene encodes a polypeptide with an altered amino acid as described in the present invention.

[0283] In addition, antibodies to the hMLH1 or hMSH2 gene and variants thereof can be raised for use in the examination of the function of the truncated transcripts of the hMLH1 or hMSH2 gene. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

[0284] Antibodies generated against the hMLH1 and hMSH2 genes of the present invention can be obtained by direct injection into an animal or by administering the gene to an animal, preferably a nonhuman. The antibody so obtained will then bind the hMLH1 or hMSH2 gene or itself. In this manner, even a fragment of the gene can be used to generate thee antibodies.

[0285] For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler et al. Nature 1975 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al. Immunology Today 1983 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp. 77-96).

[0286] Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to the hMLH1 or hMSH2 genes of this invention. Also, transgenic mice may be used to express humanized antibodies to the hMLH1 or hMSH2 genes of this invention.

[0287] The following nonlimiting examples are provided to further illustrate the present invention.

EXAMPLES Example 1 Patients and Samples

[0288] A total of 76 subjects were studied: 50 unrelated patients diagnosed with colorectal cancer at <30 years of age and 26 anonymous donors. There were 15 male and 11 female anonymous donors who were cancer free at the time of sampling and whose mean ages was 41 years. None of the study subjects were referred specifically because of a family history of colon cancer. All cancer patients had histologically confirmed colorectal cancer.

[0289] Peripheral blood was drawn from each subject and DNA was purified from peripheral-blood leukocytes.

Example 2 Genomic Sequencing

[0290] DNA was extracted from peripheral blood using the Nucleon DNA Extraction Kit, Scotlab, Lanarkshire, U.K. or using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, Minn.) as per the manufacturer's instructions. Each exon of hMSH2 and hMLH1 was amplified by PCR using 40 ng of genomic DNA in a volume of 50 μL. Final reaction concentrations were 1×PCR Buffer II (Perkin Elmer), 3.0 mM MgCl₂ (or 1.5 mM for hMSH2 exon 1), 0.2 mM dNTPs, 10 pmol of each specific oligonucleotide primer, and 1.25 units of Taq polymerase. Amplification was hot-started at 94° C. for 3 minutes, followed by 35 cycles of 94° C. for 20 seconds; 55° C. for 20 seconds; 72° C. for 40 seconds. The final reaction was extended at 72° C. for 10 minutes, followed by storage at 4° C. Cycle sequencing used the PRISM Ready Dye Terminator Cycle Sequencing kit with AmpliTaq DNA polymerase, FS (Taq-FS; Perkin Elmer/Applied Biosystems) and an Applied Biosystems DNA Sequencer model 373A or 377 (Parker et al. BioTechniques 1996 21:694-699) according to the manufacturer's instructions. DNA sequence analysis was performed using Sequencher 3.0 (Gene Codes, Ann Arbor, Mich.) software by comparing published genomic sequences of hMLH1 (Han et al. Hum. Mol. Genet. 1995 4:237-242; Kolodner et al. Cancer Res. 1995 55:242-248) and hMSH2 (Kolodner, et al. Genomics 1994 24:516-526) with that of cancer cases or of random donors.

[0291] Examples of primers used for mutations in patients 815, 830 and 1157 are as follows:

[0292] (1) Patient 815, splice error in hMLH1 exon 1:

[0293] Forward primer:

[0294] 5′-TGTAAAACGACGGCCAGTCTGAGGTGATTGGCTGAAG-3′ (SEQ ID NO: 185)

[0295] Reverse primer:

[0296] 5′-GGAAACAGCTATGACCATGCCGTTAAGTCGTAGCCCTT-3′ (SEQ ID NO: 186)

[0297] (2) Patient 830, premature stop codon in hMSH2 exon 13:

[0298] Forward primer:

[0299] 5′-TGTAAAACGACGGCCAGTCGATTAATCATCAGTGTAC-3′ (SEQ ID NO: 187)

[0300] Reverse primer:

[0301] 5′-GGAAACAGCTATGACCATGCAGAGACATACATTTCTATCTTC-3′ (SEQ ID NO: 188)

[0302] (3) Patient 1157, missense in initial ATG of hMSH2 (exon 1):

[0303] Forward primer:

[0304] 5′-TGTAAAACGACGGCCAGTCGCATTTTCTTCAACCAGGA-3′ (SEQ ID NO: 189)

[0305] Reverse primer:

[0306] 5′-GGAAACAGCTATGACCATGCCTCCCCAGCACGCGCC-3′ (SEQ ID NO: 190)

Example 2 In Vitro Synthesized-Protein-Truncation Assay (IVSP)

[0307] cDNA was generated by reverse transcription of RNA purified from lymphoblastoid cell lines from the affected index case. PCR amplification of the CDNA was used to introduce a 17-bp consensus T7 promoter sequence and a mammalian translation-initiation sequence in frame with a unique hMLH1 or hMSH2 sequence. PCR primer sequences and conditions were similar to those previously described in Example 1. Each gene was amplified in two or three overlapping segments. Resultant PCR products were used in a coupled transcription-translation reaction (Promega) incorporating 2-5 μCi of ³⁵S-methionine. Labeled in vitro-transcribed protein products from the reaction were heat denatured and were analyzed by use of 8%, 10% and 12% SDS-PAGE gels. Gels were washed in fixative and were autoradiographed overnight at room temperature. All samples showing truncated protein products were reamplified independently, and an additional IVSP analysis was performed for conformation. For each analysis, normal control samples were run in parallel, and the wild-type full length protein was noted. In most analyses, artifactual bands were visible, presumably due to internally initiations since these were visible in samples form normal controls.

Example 3 Long Range PCR

[0308] For long range PCR of the novel mutation of hMSH2 discovered in patient 817, the GeneAmp XL PCR Kit (Perkin Elmer) was used with the following primers:

[0309] Forward primer:

[0310] 5′-GGCCATTGTCACAGAGGATAAGA-3′ (SEQ ID NO: 191)

[0311] Reverse primer:

[0312] 5′-ACACAGCCCACGAAGGAGTG-3′ (SEQ ID NO: 192)

[0313] The reaction mixture contained about 400 ng of genomic DNA in a volume of 50 μL. Final reaction concentrations were 1×PCR Buffer II (Perkin Elmer), 1.5 mM Mg(OAc)₂, 0.8 mM dNTPs, 40 pmol of each specific oligonucleotide primer, and 4 units of rTth DNA polymerase. Amplification was hot-started at 94° C. for 1 minute, followed by 26 cycles of 94° C. for 15 second and 68° C. for 10 minutes. The final reaction was extended at 72° C. for 10 minutes, followed by storage at 40° C.

[0314] Replicate cDNA sequencing of samples from patient 817 reproducibly demonstrated a truncation in hMSH2 due to deletion of the entire exon 13. However, extensive genomic sequencing failed to identify the mutation at the DNA level. Hence, the intronic region around exon 13 was analyzed by long range PCR to determine whether any large genomic deletion had completely removed that exon. Forward primer was in exon 12 and reverse in exon 14, giving around 15.5 kb wild type product. Using this approach, patient 817 was shown to carry a large deletion of approximately 3 kb which resulted in removal of exon 13.

Example 4 Characterization of mutation in patient 579

[0315] Characterization of the mutation in patient 579 was more complex. Replicate hMSH2 IVSPs for patient 579 detected a very short protein fragment, which could not be explained on the basis of the His-Tyr mutation at codon 639, identified by genomic sequencing. Accordingly, additional genomic sequencing needed to be performed which resulted in identification of the second mutation at the splice acceptor site of exon 14. Using restriction-site changes induced by each mutation, both variants were traced through the family and were shown to reside on the same allele. Extensive sequencing of the reverse transcription-PCR products revealed that this complex double mutation results in an in-frame deletion of exons 12-14, thus accounting for the very short IVSP fragment. A His-Tyr mutation at codon 639 which results in a surrogate splice donor site and a 92-bp frameshift deletion of nucleotides 1914-2006, generating a premature termination codon 17 bp downstream of the exon 13 splice acceptor site has been described previously by Leach et al. Cell 1993 75:1215-1225 and Liu et al. Cancer Res. 1994 54:4590-4594. However, the 92 bp splice mutation reported to be present in this mutation was not present in patient 579, thus confirming that the double mutation in patient 579 is distinct from that reported by Liu et al. Cancer Res. 1994 54:4590-4594.

[0316] All publications including, but not limited to, patents and patent applications, cited in this specification, are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

[0317] The above description fully discloses the invention, including preferred embodiments thereof. Modifications and improvements of the embodiments specifically disclosed herein are within the scope of the following claims. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. Therefore, the examples provided herein are to be construed as merely illustrative and are not a limitation of the scope of the present invention in any way. The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows.

1 192 1 19 DNA Homo sapiens 1 aggcactgag gtgattggc 19 2 54 DNA Homo sapiens 2 tgaaggcact tccgttgagc atctagacgt ttccttggct cttctggcgc caaa 54 3 51 DNA Homo sapiens 3 ggaagttatc cagcggccag ctaatgctat caaagagatg attgagaact g 51 4 21 DNA Homo sapiens 4 gtacggaggg agtcgagccg g 21 5 19 DNA Homo sapiens 5 gctcacttaa gggctacga 19 6 8 DNA Homo sapiens 6 cttaacgg 8 7 22 DNA Homo sapiens 7 aatatgtaca ttagagtagt tg 22 8 35 DNA Homo sapiens 8 cagactgata aattattttc tgtttgattt gccag 35 9 91 DNA Homo sapiens 9 tttagatgca aaatccacaa gtattcaagt gattgttaaa gagggaggcc tgaagttgat 60 tcagatccaa gacaatggca ccgggatcag g 91 10 46 DNA Homo sapiens 10 gtaagtaaaa cctcaaagta gcaggatgtt tgtgcgcttc atggaa 46 11 19 DNA Homo sapiens 11 gagtcaggac ctttctctg 19 12 22 DNA Homo sapiens 12 agagatttgg aaaatgagta ac 22 13 34 DNA Homo sapiens 13 atgattattt actcatcttt ttggtatcta acag 34 14 99 DNA Homo sapiens 14 aaagaagatc tggatattgt atgtgaaagg ttcactacta gtaaactgca gtcctttgag 60 gatttagcca gtatttctac ctatggcttt cgaggtgag 99 15 33 DNA Homo sapiens 15 gtaagctaaa gattcaagaa atgtgtaaaa tat 33 16 19 DNA Homo sapiens 16 cctcctgtga tgacattgt 19 17 20 DNA Homo sapiens 17 aacctttccc tttggtgagg 20 18 59 DNA Homo sapiens 18 tgacagtggg tgacccagca gtgagttttt ctttcagtct attttctttt cttccttag 59 19 74 DNA Homo sapiens 19 gctttggcca gcataagcca tgtggctcat gttactatta caacgaaaac agctgatgga 60 aagtgtgcat acag 74 20 53 DNA Homo sapiens 20 gtatagtgct gacttctttt actcatatat attcattctg aaatgtattt tgg 53 21 20 DNA Homo sapiens 21 gcctaggtct cagagtaatc 20 22 7 DNA Homo sapiens 22 ttgatat 7 23 22 DNA Homo sapiens 23 gattttctct tttccccttg gg 22 24 51 DNA Homo sapiens 24 attagtatct atctctctac tggatattaa tttgttatat tttctcatta g 51 25 73 DNA Homo sapiens 25 agcaagttac tcagatggaa aactgaaagc ccctcctaaa ccatgtgctg gcaatcaagg 60 gacccagatc acg 73 26 21 DNA Homo sapiens 26 gtaagaatgg tacatgggac a 21 27 23 DNA Homo sapiens 27 gtaaattgtt gaagctttgt ttg 23 28 26 DNA Homo sapiens 28 gggttttatt ttcaagtact tctatg 26 29 31 DNA Homo sapiens 29 aatttacaag aaaaatcaat cttctgttca g 31 30 92 DNA Homo sapiens 30 gtggaggacc ttttttacaa catagccacg aggagaaaag ctttaaaaaa tccaagtgaa 60 gaatatggga aaattttgga agttgttggc ag 92 31 61 DNA Homo sapiens 31 gtacagtcca aaatctggga gtgggtctct gagatttgtc atcaaagtaa tgtgttctag 60 t 61 32 26 DNA Homo sapiens 32 gctcatacat tgaacagttg ctgagc 26 33 18 DNA Homo sapiens 33 ctagtgtgtg tttttggc 18 34 35 DNA Homo sapiens 34 aactcttttc ttactctttt gtttttcttt tccag 35 35 43 DNA Homo sapiens 35 gtattcagta cacaatgcag gcattagttt ctcagttaaa aaa 43 36 70 DNA Homo sapiens 36 gtaagttctt ggtttatggg ggatggtttt gttttatgaa aagaaaaaag gggattttta 60 atagtttgct 70 37 18 DNA Homo sapiens 37 ggtggagata aggttatg 18 38 23 DNA Homo sapiens 38 ctcagccatg agacaataaa tcc 23 39 27 DNA Homo sapiens 39 ttgtgtcttc tgctgtttgt ttatcag 27 40 89 DNA Homo sapiens 40 caaggagaga cagtagctga tgttaggaca ctacccaatg cctcaaccgt ggacaatatt 60 cgctccatct ttggaaatgc tgttagtcg 89 41 57 DNA Homo sapiens 41 gtatgtcgat aacctatata aaaaaatctt ttacatttat tatcttggtt tatcatt 57 42 21 DNA Homo sapiens 42 ccatcacatt atttgggaac c 21 43 18 DNA Homo sapiens 43 caaaagcttc agaatctc 18 44 11 DNA Homo sapiens 44 ttttctaata g 11 45 113 DNA Homo sapiens 45 agaactgata gaaattggat gtgaggataa aaccctagcc ttcaaaatga atggttacat 60 atccaatgca aactactcag tgaagaagtg catcttctta ctcttcatca acc 113 46 28 DNA Homo sapiens 46 gtaagttaaa aagaaccaca tgggaaat 28 47 23 DNA Homo sapiens 47 ccactcacag gaaacaccca cag 23 48 24 DNA Homo sapiens 48 catgactttg tgtgaatgta cacc 24 49 56 DNA Homo sapiens 49 tgtgacctca cccctcagga cagttttgaa ctggttgctt tctttttatt gtttag 56 50 94 DNA Homo sapiens 50 atcgtctggt agaatcaact tccttgagaa aagccataga aacagtgtat gcagcctatt 60 tgcccaaaaa cacacaccca ttcctgtacc tcag 94 51 41 DNA Homo sapiens 51 gtaatgtagc accaaactcc tcaaccaaga ctcacaagga a 41 52 24 DNA Homo sapiens 52 cagatgttct atcaggctct cctc 24 53 20 DNA Homo sapiens 53 gggctttttc tccccctccc 20 54 39 DNA Homo sapiens 54 actatctaag gtaattgttc tctcttattt tcctgacag 39 55 154 DNA Homo sapiens 55 tttagaaatc agtccccaga atgtggatgt taatgtgcac cccacaaagc atgaagttca 60 cttcctgcac gaggagagca tcctggagcg ggtgcagcag cacatcgaga gcaagctcct 120 gggctccaat tcctccagga tgtacttcac ccag 154 56 58 DNA Homo sapiens 56 gtcagggcgc ttctcatcca gctacttctc tggggccttt gaaatgtgcc cggccaga 58 57 18 DNA Homo sapiens 57 cgtgagagcc cagatttt 18 58 19 DNA Homo sapiens 58 aattatacct catactagc 19 59 116 DNA Homo sapiens 59 ttctttctta gtactgctcc atttggggac ctgtatatct atacttctta ttctgagtct 60 ctccactata tatatatata tatatatatt tttttttttt ttttttttta atacag 116 60 371 DNA Homo sapiens 60 actttgctac caggacttgc tggcccctct ggggagatgg ttaaatccac aacaagtctg 60 acctcgtctt ctacttctgg aagtagtgat aaggtctatg cccaccagat ggttcgtaca 120 gattcccggg aacagaagct tgatgcattt ctgcagcctc tgagcaaacc cctgtccagt 180 cagccccagg ccattgtcac agaggataag acagatattt ctagtggcag ggctaggcag 240 caagatgagg agatgcttga actcccagcc cctgctgaag tggctgccaa aaatcagagc 300 ttggaggggg atacaacaaa ggggacttca gaaatgtcag agaagagagg acctacttcc 360 agcaacccca g 371 61 29 DNA Homo sapiens 61 gtatggcctt ttgggaaaag tacagccta 29 62 23 DNA Homo sapiens 62 cctcctttat tctgtaataa aac 23 63 20 DNA Homo sapiens 63 tgcaacccac aaaatttggc 20 64 53 DNA Homo sapiens 64 taagtttaaa aacaagaata ataatgatct gcacttcctt ttcttcattg cag 53 65 149 DNA Homo sapiens 65 aaagagacat cgggaagatt ctgatgtgga aatggtggaa gatgattccc gaaaggaaat 60 gactgcagct tgtacccccc ggagaaggat cattaacctc actagtgttt tgagtctcca 120 ggaagaaatt aatgagcagg gacatgagg 149 66 50 DNA Homo sapiens 66 gtacgtaaac gctgtggcct gcctgggatg catagggcct caactgccaa 50 67 20 DNA Homo sapiens 67 ggttttggaa atggagaaag 20 68 18 DNA Homo sapiens 68 tggtgtctct agttctgg 18 69 82 DNA Homo sapiens 69 tgcctggtgc tttggtcaat gaagtggggt tggtaggatt ctattactta cctgtttttt 60 ggttttattt tttgttttgc ag 82 70 109 DNA Homo sapiens 70 ttctccggga gatgttgcat aaccactcct tcgtgggctg tgtgaatcct cagtgggcct 60 tggcacagca tcaaaccaag ttataccttc tcaacaccac caagcttag 109 71 25 DNA Homo sapiens 71 gtaaatcagc tgagtgtgtg aacaa 25 72 20 DNA Homo sapiens 72 gcagagctac tacaacaatg 20 73 18 DNA Homo sapiens 73 cccatttgtc ccaactgg 18 74 57 DNA Homo sapiens 74 ttgtatctca agcatgaatt cagcttttcc ttaaagtcac ttcattttta ttttcag 57 75 64 DNA Homo sapiens 75 tgaagaactg ttctaccaga tactcattta tgattttgcc aattttggtg ttctcaggtt 60 atcg 64 76 23 DNA Homo sapiens 76 gtaagtttag atccttttca ctt 23 77 19 DNA Homo sapiens 77 ctgacatttc aactgaccg 19 78 22 DNA Homo sapiens 78 catttggatg ctccgttaaa gc 22 79 30 DNA Homo sapiens 79 ttgctccttc atgttcttgc ttcttcctag 30 80 165 DNA Homo sapiens 80 gagccagcac cgctctttga ccttgccatg cttgccttag atagtccaga gagtggctgg 60 acagaggaag atggtcccaa agaaggactt gctgaataca ttgttgagtt tctgaagaag 120 aaggctgaga tgcttgcaga ctatttctct ttggaaattg atgag 165 81 36 DNA Homo sapiens 81 gtgtgacagc cattcttata cttctgttgt attctc 36 82 23 DNA Homo sapiens 82 caaataaaat ttccagccgg gtg 23 83 22 DNA Homo sapiens 83 ggaaaggcac tggagaaatg gg 22 84 59 DNA Homo sapiens 84 atttgtttaa actatgacag cattatttct tgttcccttg tcctttttcc tgcaagcag 59 85 93 DNA Homo sapiens 85 gaagggaacc tgattggatt accccttctg attgacaact atgtgccccc tttggaggga 60 ctgcctatct tcattcttcg actagccact gag 93 86 29 DNA Homo sapiens 86 gtcagtgatc aagcagatac taagcattt 29 87 25 DNA Homo sapiens 87 cggtacatgc atgtgtgctg gaggg 25 88 20 DNA Homo sapiens 88 taagtagtct gtgatctccg 20 89 60 DNA Homo sapiens 89 tttagaatga gaatgtttaa attcgtacct attttgaggt attgaatttc tttggaccag 60 90 114 DNA Homo sapiens 90 gtgaattggg acgaagaaaa ggaatgtttt gaaagcctca gtaaagaatg cgctatgttc 60 tattccatcc ggaagcagta catatctgag gagtcgaccc tctcaggcca gcag 114 91 35 DNA Homo sapiens 91 gtacagtggt gatgcacact ggcaccccag gacta 35 92 18 DNA Homo sapiens 92 ggacaggacc tcatacat 18 93 18 DNA Homo sapiens 93 gacaccagtg tatgttgg 18 94 42 DNA Homo sapiens 94 gatgcaaaca gggaggctta tgacatctaa tgtgttttcc ag 42 95 168 DNA Homo sapiens 95 agtgaagtgc ctggctccat tccaaactcc tggaagtgga ctgtggaaca cattgtctat 60 aaagccttgc gctcacacat tctgcctcct aaacatttca cagaagatgg aaatatcctg 120 cagcttgcta acctgcctga tctatacaaa gtctttgaga ggtgttaa 168 96 21 DNA Homo sapiens 96 atatggttat ttatgcactg t 21 97 20 DNA Homo sapiens 97 gggatgtgtt cttctttctc 20 98 60 DNA Homo sapiens 98 tgtattccga tacaaagtgt tgtatcaaag tgtgatatac aaagtgtacc aacataagtg 60 99 31 DNA Homo sapiens 99 ggcgggaaac agcttagtgg gtgtggggtc g 31 100 20 DNA Homo sapiens 100 cgcattttct tcaaccagga 20 101 17 DNA Homo sapiens 101 ggtgaggagg tttcgac 17 102 211 DNA Homo sapiens 102 atggcggtgc agccgaagga gacgctgcag ttggagagcg cggccgaggt cggcttcgtg 60 cgcttctttc agggcatgcc ggagaagccg accaccacag tgcgcctttt cgaccggggc 120 gacttctata cggcgcacgg cgaggacgcg ctgctggccg cccgggaggt gttcaagacc 180 cagggggtga tcaagtacat ggggccggca g 211 103 14 DNA Homo sapiens 103 gtgagggccg ggac 14 104 17 DNA Homo sapiens 104 ggcgcgtgct ggggagg 17 105 21 DNA Homo sapiens 105 gtccagctaa tacagtgctt g 21 106 46 DNA Homo sapiens 106 aacatgtaat atctcaaatc tgtaatgtac tttttttttt tttaag 46 107 155 DNA Homo sapiens 107 gagcaaagaa tctgcagagt gttgtgctta gtaaaatgaa ttttgaatct tttgtaaaag 60 atcttcttct ggttcgtcag tatagagttg aagtttataa gaatagagct ggaaataagg 120 catccaagga gaatgattgg tatttggcat ataag 155 108 33 DNA Homo sapiens 108 gtaattatct tcctttttaa tttacttatt ttt 33 109 25 DNA Homo sapiens 109 ttaagagtag aaaaataaaa atgtg 25 110 32 DNA Homo sapiens 110 attaataagg ttcatagagt ttggattttt cc 32 111 26 DNA Homo sapiens 111 ttataaaatt ttaaagtatg ttcaag 26 112 36 DNA Homo sapiens 112 agtttgttaa atttttaaaa ttttattttt acttag 36 113 279 DNA Homo sapiens 113 gcttctcctg gcaatctctc tcagtttgaa gacattctct ttggtaacaa tgatatgtca 60 gcttccattg gtgttgtggg tgttaaaatg tccgcagttg atggccagag acaggttgga 120 gttgggtatg tggattccat acagaggaaa ctaggactgt gtgaattccc tgataatgat 180 cagttctcca atcttgaggc tctcctcatc cagattggac caaaggaatg tgttttaccc 240 ggaggagaga ctgctggaga catggggaaa ctgagacag 279 114 23 DNA Homo sapiens 114 gtaagcaaat tgagtctagt gat 23 115 25 DNA Homo sapiens 115 agaggagatt ccaggcctag gaaag 25 116 21 DNA Homo sapiens 116 tctttaattg acatgatact g 21 117 24 DNA Homo sapiens 117 tttttgcttt tcttattcct tttc 24 118 33 DNA Homo sapiens 118 tcatagtagt ttaaactatt tctttcaaaa tag 33 119 147 DNA Homo sapiens 119 ataattcaaa gaggaggaat tctgatcaca gaaagaaaaa aagctgactt ttccacaaaa 60 gacatttatc aggacctcaa ccggttgttg aaaggcaaaa agggagagca gatgaatagt 120 gctgtattgc cagaaatgga gaatcag 147 120 85 DNA Homo sapiens 120 gtacatggat tataaatgtg aattacaata tatataatgt aaatatgtaa tatataataa 60 ataatatgta aactatagtg acttt 85 121 20 DNA Homo sapiens 121 ttagaaggat atttctgtca 20 122 10 DNA Homo sapiens 122 actggcacca 10 123 24 DNA Homo sapiens 123 gtggtataga aatcttcgat tttt 24 124 16 DNA Homo sapiens 124 aaattcttaa ttttag 16 125 150 DNA Homo sapiens 125 gttgcagttt catcactgtc tgcggtaatc aagtttttag aactcttatc agatgattcc 60 aactttggac agtttgaact gactactttt gacttcagcc agtatatgaa attggatatt 120 gcagcagtca gagcccttaa cctttttcag 150 126 23 DNA Homo sapiens 126 gtaaaaaaaa aaaaaaaaaa aaa 23 127 22 DNA Homo sapiens 127 aaaagggtta aaaatgttga tt 22 128 13 DNA Homo sapiens 128 ttaaaaaatg ttt 13 129 23 DNA Homo sapiens 129 cattgacata tactgaagaa gct 23 130 27 DNA Homo sapiens 130 tataaaggag ctaaaatatt tggaaat 27 131 43 DNA Homo sapiens 131 attatacttg gattagataa ctagctttaa atgggtgtat ttt 43 132 22 DNA Homo sapiens 132 ttcactaatg agcttgccat tc 22 133 26 DNA Homo sapiens 133 tttctatttt attttttgtt tactag 26 134 134 DNA Homo sapiens 134 ggttctgttg aagataccac tggctctcag tctctggctg ccttgctgaa taagtgtaaa 60 acccctcaag gacaaagact tgttaaccag tggattaagc agcctctcat ggataagaac 120 agaatagagg agag 134 135 44 DNA Homo sapiens 135 gtatgttatt agtttatact ttcgttagtt ttatgtaacc tgca 44 136 20 DNA Homo sapiens 136 gttacccaca tgattatacc 20 137 20 DNA Homo sapiens 137 cttacgtgct tagttgataa 20 138 53 DNA Homo sapiens 138 attttaattt tatactaaaa tattttacat taattcaagt taatttattt cag 53 139 200 DNA Homo sapiens 139 attgaattta gtggaagctt ttgtagaaga tgcagaattg aggcagactt tacaagaaga 60 tttacttcgt cgattcccag atcttaaccg acttgccaag aagtttcaaa gacaagcagc 120 aaacttacaa gattgttacc gactctatca gggtataaat caactaccta atgttataca 180 ggctctggaa aaacatgaag 200 140 24 DNA Homo sapiens 140 gtaacaagtg attttgtttt tttg 24 141 24 DNA Homo sapiens 141 ttttccttca actcatacaa tata 24 142 26 DNA Homo sapiens 142 tttgtattct gtaaaatgag atcttt 26 143 30 DNA Homo sapiens 143 ttatttgttt gttttactac tttcttttag 30 144 110 DNA Homo sapiens 144 gaaaacacca gaaattattg ttggcagttt ttgtgactcc tcttactgat cttcgttctg 60 acttctccaa gtttcaggaa atgatagaaa caactttaga tatggatcag 110 145 26 DNA Homo sapiens 145 gtatgcaata tactttttaa tttaag 26 146 25 DNA Homo sapiens 146 cagtagttat ttttaaaaag caaag 25 147 23 DNA Homo sapiens 147 ctttacccat tatttatagg att 23 148 23 DNA Homo sapiens 148 ttgtcacttt gttctgtttg cag 23 149 124 DNA Homo sapiens 149 gtggaaaacc atgaattcct tgtaaaacct tcatttgatc ctaatctcag tgaattaaga 60 gaaataatga atgacttgga aaagaagatg cagtcaacat taataagtgc agccagagat 120 cttg 124 150 20 DNA Homo sapiens 150 gtaagaatgg gtcattggag 20 151 23 DNA Homo sapiens 151 gttggaataa ttcttttgtc tat 23 152 25 DNA Homo sapiens 152 tagtaggtat ttatggaata ctttt 25 153 22 DNA Homo sapiens 153 tcttttcttc ttgtttatca ag 22 154 151 DNA Homo sapiens 154 gcttggaccc tggcaaacag attaaactgg attccagtgc acagtttgga tattactttc 60 gtgtaacctg taaggaagaa aaagtccttc gtaacaataa aaactttagt actgtagata 120 tccagaagaa tggtgttaaa tttaccaaca g 151 155 36 DNA Homo sapiens 155 gtttgtaagt cattattata tttttaaccc tttatt 36 156 21 DNA Homo sapiens 156 aattccctaa atgctctaac a 21 157 21 DNA Homo sapiens 157 cattgcttct agtacacatt t 21 158 36 DNA Homo sapiens 158 taatattttt aataaaactg ttatttcgat ttgcag 36 159 98 DNA Homo sapiens 159 caaattgact tctttaaatg aagagtatac caaaaataaa acagaatatg aagaagccca 60 ggatgccatt gttaaagaaa ttgtcaatat ttcttcag 98 160 19 DNA Homo sapiens 160 gtaaacttaa tagaactaa 19 161 21 DNA Homo sapiens 161 taatgttctg aatgtcacct g 21 162 21 DNA Homo sapiens 162 tcagtattcc tgtgtacatt t 21 163 21 DNA Homo sapiens 163 tctgttttta tttttataca g 21 164 246 DNA Homo sapiens 164 gctatgtaga accaatgcag acactcaatg atgtgttagc tcagctagat gctgttgtca 60 gctttgctca cgtgtcaaat ggagcacctg ttccatatgt acgaccagcc attttggaga 120 aaggacaagg aagaattata ttaaaagcat ccaggcatgc ttgtgttgaa gttcaagatg 180 aaattgcatt tattcctaat gacgtatact ttgaaaaaga taaacagatg ttccacatca 240 ttactg 246 165 16 DNA Homo sapiens 165 gtaaaaaacc tggttt 16 166 19 DNA Homo sapiens 166 ttgggctttg tgggggtaa 19 167 19 DNA Homo sapiens 167 cgattaatca tcagtgtac 19 168 78 DNA Homo sapiens 168 agtttaggac taacaatcca tttattagta gcagaaagaa gtttaaaatc ttgctttctg 60 atataatttg ttttgtag 78 169 205 DNA Homo sapiens 169 gccccaatat gggaggtaaa tcaacatata ttcgacaaac tggggtgata gtactcatgg 60 cccaaattgg gtgttttgtg ccatgtgagt cagcagaagt gtccattgtg gactgcatct 120 tagcccgagt aggggctggt gacagtcaat tgaaaggagt ctccacgttc atggctgaaa 180 tgttggaaac tgcttctatc ctcag 205 170 23 DNA Homo sapiens 170 gtaagtgcat ctcctagtcc ctt 23 171 23 DNA Homo sapiens 171 gaagatagaa atgtatgtct ctg 23 172 21 DNA Homo sapiens 172 ccacatttta tgtgatggga a 21 173 24 DNA Homo sapiens 173 atttcatgta attatgtgct tcag 24 174 248 DNA Homo sapiens 174 gtctgcaacc aaagattcat taataatcat agatgaattg ggaagaggaa cttctaccta 60 cgatggattt gggttagcat gggctatatc agaatacatt gcaacaaaga ttggtgcttt 120 ttgcatgttt gcaacccatt ttcatgaact tactgccttg gccaatcaga taccaactgt 180 taataatcta catgtcacag cactcaccac tgaagagacc ttaactatgc tttatcaggt 240 gaagaaag 248 175 56 DNA Homo sapiens 175 gtatgtacta ttggagtact ctaaattcag aacttggtaa tgggaaactt actacc 56 176 19 DNA Homo sapiens 176 cttctcatgc tgtcccctc 19 177 23 DNA Homo sapiens 177 acgcttcccc aaatttctta tag 23 178 176 DNA Homo sapiens 178 gtgtctgtga tcaaagtttt gggattcatg ttgcagagct tgctaatttc cctaagcatg 60 taatagagtg tgctaaacag aaagccctgg aacttgagga gtttcagtat attggagaat 120 cgcaaggata tgatatcatg gaaccagcag caaagaagtg ctatctggaa agagag 176 179 17 DNA Homo sapiens 179 gtttgtcagt ttgtttt 17 180 21 DNA Homo sapiens 180 catagtttaa cttagcttct c 21 181 21 DNA Homo sapiens 181 attactcatg ggacattcac a 21 182 10 DNA Homo sapiens 182 tgtgtttcag 10 183 171 DNA Homo sapiens 183 caaggtgaaa aaattattca ggagttcctg tccaaggtga aacaaatgcc ctttactgaa 60 atgtcagaag aaaacatcac aataaagtta aaacagctaa aagctgaagt aatagcaaag 120 aataatagct ttgtaaatga aatcatttca cgaataaaag ttactacgtg a 171 184 20 DNA Homo sapiens 184 atcccagtaa tggaatgaag 20 185 37 DNA Artificial Sequence Description of Artificial Sequence Synthetic 185 tgtaaaacga cggccagtct gaggtgattg gctgaag 37 186 38 DNA Artificial Sequence Description of Artificial Sequence Synthetic 186 ggaaacagct atgaccatgc cgttaagtcg tagccctt 38 187 37 DNA Artificial Sequence Description of Artificial Sequence Synthetic 187 tgtaaaacga cggccagtcg attaatcatc agtgtac 37 188 42 DNA Artificial Sequence Description of Artificial Sequence Synthetic 188 ggaaacagct atgaccatgc agagacatac atttctatct tc 42 189 38 DNA Artificial Sequence Description of Artificial Sequence Synthetic 189 tgtaaaacga cggccagtcg cattttcttc aaccagga 38 190 36 DNA Artificial Sequence Description of Artificial Sequence Synthetic 190 ggaaacagct atgaccatgc ctccccagca cgcgcc 36 191 23 DNA Artificial Sequence Description of Artificial Sequence Synthetic 191 ggccattgtc acagaggata aga 23 192 20 DNA Artificial Sequence Description of Artificial Sequence Synthetic 192 acacagccca cgaaggagtg 20 

What is claimed is:
 1. A variant human MLH1 or MSH2 gene comprising hMLH1 mutant 1, hMLH1 mutant 2, hMSH2 mutant 1, hMSH2 mutant 2 or hMSH2 mutant
 3. 2. A method of diagnosing hereditary non-polyposis colorectal cancer in a patient comprising: (a) obtaining a DNA sample from a patient; and (b) screening the DNA sample for the variant human MLH1 or MSH2 gene of claim 1 , wherein the presence of the variant gene is indicative of hereditary non-polyposis colorectal cancer.
 3. A method for predicting susceptibility of a patient to developing hereditary non-polyposis colorectal cancer comprising: (a) obtaining a DNA sample from a patient; and (b) screening the DNA sample for the variant human MLH1 or MSH2 gene of claim 1 , wherein the presence of the variant gene is indicative of a susceptibility to hereditary non-polyposis colorectal cancer.
 4. A method of identifying mutants in splice donor or acceptor sites of a human MLH1 gene comprising sequencing splice donor or acceptor sites of the human MLH1 gene with intronic primers for the human MLH1 gene and analyzing the sequences to identify any mutants.
 5. An intronic primer for human MLH1.
 6. A method of identifying mutants in splice donor or acceptor sites of a human MSH2 gene, comprising sequencing splice donor or acceptor sites of the human MSH2 gene with intronic primers for the human MSH2 gene and analyzing the sequences to identify any mutants.
 7. An intronic primer for human MSH2.
 8. A transgenic model system for colorectal cancer comprising cells expressing the variant human MLH1 or MSH2 gene of claim 1 . 